This project will examine the mechanisms and sites of action of divalent cations that regulate the behavior of calcium (Ca2+)- and voltage-activated BK-type Slo1 potassium channels. The goal of the experiments is to define the multiple sites at which Ca2+ and Mg2+ act on Slo1 channels and the mechanism by which these sites may regulate channel function. These goals will be met through the concerted use of biophysical, biochemical, and molecular biological approaches. Previous work has shown that Slo1 channel function is regulated by at least two divalent cation sites with differing affinities and a powerful mechanistic framework for analyzing these allosteric effects has been developed. To define the locations and functional roles of these sites, the homologous Slo3 variant which lacks the key divalent cation regulatory sites will be employed. Chimeric constructs of Slo1/Slo3 subunits will be examined to determine regions that confer low and high affinity regulation by Ca2+ and Mg2+. These results will be analyzed in terms of allosteric models that allow definition of the presence of particular binding sites and the affinity of those sites for divalent cations. Complementary biochemical work will also be done on fusion peptides obtained from portions of the Slo1 subunit to demonstrate whether particular regions may specifically bind Ca2+ and/or Mg2+. The proposed work builds on previous work from this lab, and is expected to lead to insights into the fundamental mechanisms by which binding of Ca2+ and other divalent cations to a protein can regulate the functional properties of that protein. Because of the ubiquitous importance of Ca2+ as an intracellular messenger, understanding how Ca2+ concentrations spanning over four orders of magnitude can regulate the function of the Slo1 channel protein is of importance to the general question of how Ca2+-sensing proteins detect and respond to Ca2+.